Our goal is to determine the mechanism by which the highly replicated coronary artery disease (CAD) association at 6q23.2 contributes to disease risk. We have made significant progress toward this goal, demonstrating that: i) functional variation at this locus regulates expression of TCF21 by altering growth factor and developmental pathway transcriptional signaling, as well as post-transcriptional miRNA binding; ii) Tcf21 is expressed in the arterial adventitia and a subset of cells in the media that express low levels of smooth muscle cell (SMC) lineage markers; iii) TCF21 regulates a large number of other CAD associated genes, defining a CAD transcriptional network predicted to regulate growth factor signaling, matrix interaction, and smooth muscle processes; iv) TCF21 promotes cellular proliferation and migration, and inhibits SMC contractile marker gene expression in vitro in human coronary artery SMC; and v) lineage traced Tcf21 expressing cells migrate through the forming plaque and contribute to the SMC lineage at the fibrous cap. Taken together, these data suggest the Central Hypothesis for this proposal: TCF21 regulates fundamental transcription pathways to mediate phenotypic modulation of medial SMC, promoting their dedifferentiation, expansion and migration to the fibrous cap where they again adopt the SMC lineage and contribute to cap integrity and stability. Perturbation of TCF21 expression disrupts this process, increasing the risk of plaque rupture. This hypothesis is also supported by TCF21's known role in development where it mediates expansion of SMC precursor cells of the epicardium and promotes epithelial-mesenchymal transition as these cells migrate into the myocardium and differentiate into coronary SMC. Further studies proposed in this renewal application will utilize genetic mouse models and other reagents and datasets generated in the previous funding period, and will focus on the mechanisms by which TCF21 affects SMC fate and how perturbation of its expression leads to risk for CAD. Specifically, in Specific Aim 1 we propose to use atherosclerosis and plaque rupture models to determine how changes in Tcf21 expression alter the SMC makeup and anatomy of the fibrous cap, thus modifying the risk of plaque rupture. Specific Aim 2 will investigate the gene regulatory program of Tcf21 in vivo in mouse atherosclerosis as modulated SMC migrate through the plaque and differentiate at the fibrous cap. Specific Aim 3 will investigate the interaction of TCF21 with known molecular pathways that regulate SMC differentiation state, including the serum response factor-myocardin and the Hippo signaling pathways. Taken together, these studies will significantly advance our understanding of how SMC related processes are regulated by TCF21, and how alteration of these functions contribute to CAD risk.